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Active site remodelling accompanies thioester bond formation in the SUMO E1.

Olsen SK, Capili AD, Lu X, Tan DS, Lima CD - Nature (2010)

Bottom Line: These structures show that side chain contacts to ATP.Mg are released after adenylation to facilitate a 130 degree rotation of the Cys domain during thioester bond formation that is accompanied by remodelling of key structural elements including the helix that contains the E1 catalytic cysteine, the crossover and re-entry loops, and refolding of two helices that are required for adenylation.These changes displace side chains required for adenylation with side chains required for thioester bond formation.Mutational and biochemical analyses indicate these mechanisms are conserved in other E1s.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology, Sloan-Kettering Institute, New York, New York 10065, USA.

ABSTRACT
E1 enzymes activate ubiquitin (Ub) and ubiquitin-like (Ubl) proteins in two steps by carboxy-terminal adenylation and thioester bond formation to a conserved catalytic cysteine in the E1 Cys domain. The structural basis for these intermediates remains unknown. Here we report crystal structures for human SUMO E1 in complex with SUMO adenylate and tetrahedral intermediate analogues at 2.45 and 2.6 A, respectively. These structures show that side chain contacts to ATP.Mg are released after adenylation to facilitate a 130 degree rotation of the Cys domain during thioester bond formation that is accompanied by remodelling of key structural elements including the helix that contains the E1 catalytic cysteine, the crossover and re-entry loops, and refolding of two helices that are required for adenylation. These changes displace side chains required for adenylation with side chains required for thioester bond formation. Mutational and biochemical analyses indicate these mechanisms are conserved in other E1s.

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Analogs of the Ub/Ubl-adenylate and E1~Ub/Ubl tetrahedral intermediatesChemical structures of a, Ub/Ubl-adenylate (top) and Ub-AMSN adenylate analog12 (bottom), b, the E1~Ub/Ubl tetrahedral intermediate (top) during thioester bond formation, the Ub/Ubl-AVSN adduct12 (middle), and the E1~Ub/Ubl-AVSN tetrahedral intermediate analog (bottom). Red atoms indicate modifications in AMSN and AVSN that deviate from AMP. c, DTT sensitivity of Ub/Ubl thioester and thioether adducts for human and S. cerevisiae SUMO E1s and S. pombe ubiquitin E1. d, Cross-linking assay and time course for SUMO1-AVSN, S. cerevisiae SMT3-AVSN, and S. pombe Ub-AVSN adducts to their cognate E1s. See Methods for assay conditions.
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Figure 1: Analogs of the Ub/Ubl-adenylate and E1~Ub/Ubl tetrahedral intermediatesChemical structures of a, Ub/Ubl-adenylate (top) and Ub-AMSN adenylate analog12 (bottom), b, the E1~Ub/Ubl tetrahedral intermediate (top) during thioester bond formation, the Ub/Ubl-AVSN adduct12 (middle), and the E1~Ub/Ubl-AVSN tetrahedral intermediate analog (bottom). Red atoms indicate modifications in AMSN and AVSN that deviate from AMP. c, DTT sensitivity of Ub/Ubl thioester and thioether adducts for human and S. cerevisiae SUMO E1s and S. pombe ubiquitin E1. d, Cross-linking assay and time course for SUMO1-AVSN, S. cerevisiae SMT3-AVSN, and S. pombe Ub-AVSN adducts to their cognate E1s. See Methods for assay conditions.

Mentions: A non-hydrolyzable mimic of the acyl-adenylate intermediate (AMSN) was made by linking a cysteylglycylglycyl tripeptide to 5′-(sulfamoylaminodeoxy)adenosine (CGG-AMSN)12. To trap a covalent adduct with the E1 active site cysteine, we synthesized a 5′-(vinylsulfonylaminodeoxy)adenosine tripeptide variant (CGG-AVSN)12 containing an electrophilic center at the position predicted to be attacked by the E1 active site cysteine during thioester bond formation. These compounds were then coupled to SUMO or ubiquitin, which lacked the corresponding three C-terminal amino acids, via intein-mediated ligation13 to generate human SUMO1-AMSN and SUMO1-AVSN, S. cerevisiae SMT3-AVSN, and ubiquitin-AVSN (Fig. 1a,b; Methods).


Active site remodelling accompanies thioester bond formation in the SUMO E1.

Olsen SK, Capili AD, Lu X, Tan DS, Lima CD - Nature (2010)

Analogs of the Ub/Ubl-adenylate and E1~Ub/Ubl tetrahedral intermediatesChemical structures of a, Ub/Ubl-adenylate (top) and Ub-AMSN adenylate analog12 (bottom), b, the E1~Ub/Ubl tetrahedral intermediate (top) during thioester bond formation, the Ub/Ubl-AVSN adduct12 (middle), and the E1~Ub/Ubl-AVSN tetrahedral intermediate analog (bottom). Red atoms indicate modifications in AMSN and AVSN that deviate from AMP. c, DTT sensitivity of Ub/Ubl thioester and thioether adducts for human and S. cerevisiae SUMO E1s and S. pombe ubiquitin E1. d, Cross-linking assay and time course for SUMO1-AVSN, S. cerevisiae SMT3-AVSN, and S. pombe Ub-AVSN adducts to their cognate E1s. See Methods for assay conditions.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2866016&req=5

Figure 1: Analogs of the Ub/Ubl-adenylate and E1~Ub/Ubl tetrahedral intermediatesChemical structures of a, Ub/Ubl-adenylate (top) and Ub-AMSN adenylate analog12 (bottom), b, the E1~Ub/Ubl tetrahedral intermediate (top) during thioester bond formation, the Ub/Ubl-AVSN adduct12 (middle), and the E1~Ub/Ubl-AVSN tetrahedral intermediate analog (bottom). Red atoms indicate modifications in AMSN and AVSN that deviate from AMP. c, DTT sensitivity of Ub/Ubl thioester and thioether adducts for human and S. cerevisiae SUMO E1s and S. pombe ubiquitin E1. d, Cross-linking assay and time course for SUMO1-AVSN, S. cerevisiae SMT3-AVSN, and S. pombe Ub-AVSN adducts to their cognate E1s. See Methods for assay conditions.
Mentions: A non-hydrolyzable mimic of the acyl-adenylate intermediate (AMSN) was made by linking a cysteylglycylglycyl tripeptide to 5′-(sulfamoylaminodeoxy)adenosine (CGG-AMSN)12. To trap a covalent adduct with the E1 active site cysteine, we synthesized a 5′-(vinylsulfonylaminodeoxy)adenosine tripeptide variant (CGG-AVSN)12 containing an electrophilic center at the position predicted to be attacked by the E1 active site cysteine during thioester bond formation. These compounds were then coupled to SUMO or ubiquitin, which lacked the corresponding three C-terminal amino acids, via intein-mediated ligation13 to generate human SUMO1-AMSN and SUMO1-AVSN, S. cerevisiae SMT3-AVSN, and ubiquitin-AVSN (Fig. 1a,b; Methods).

Bottom Line: These structures show that side chain contacts to ATP.Mg are released after adenylation to facilitate a 130 degree rotation of the Cys domain during thioester bond formation that is accompanied by remodelling of key structural elements including the helix that contains the E1 catalytic cysteine, the crossover and re-entry loops, and refolding of two helices that are required for adenylation.These changes displace side chains required for adenylation with side chains required for thioester bond formation.Mutational and biochemical analyses indicate these mechanisms are conserved in other E1s.

View Article: PubMed Central - PubMed

Affiliation: Structural Biology, Sloan-Kettering Institute, New York, New York 10065, USA.

ABSTRACT
E1 enzymes activate ubiquitin (Ub) and ubiquitin-like (Ubl) proteins in two steps by carboxy-terminal adenylation and thioester bond formation to a conserved catalytic cysteine in the E1 Cys domain. The structural basis for these intermediates remains unknown. Here we report crystal structures for human SUMO E1 in complex with SUMO adenylate and tetrahedral intermediate analogues at 2.45 and 2.6 A, respectively. These structures show that side chain contacts to ATP.Mg are released after adenylation to facilitate a 130 degree rotation of the Cys domain during thioester bond formation that is accompanied by remodelling of key structural elements including the helix that contains the E1 catalytic cysteine, the crossover and re-entry loops, and refolding of two helices that are required for adenylation. These changes displace side chains required for adenylation with side chains required for thioester bond formation. Mutational and biochemical analyses indicate these mechanisms are conserved in other E1s.

Show MeSH